Protein regulation is ubiquitous in biology. It plays an essential role in controlling and coordinating a variety of cellular processes including replication, division and growth. Protein mis-regulation has been implicated in many human diseases. The emergence of new regulatory strategies often requires protein conformational heterogeneity. For example, allostery requires the ability to toggle between two (or more) distinct polypeptide structures. Similarly, a protein's ability to interact with multiple binding partners often requires sampling a range of conformations. Past experimental studies of individual polypeptides have yielded a molecular level understanding of many conformation-dependent regulatory processes. Despite this fact, we know little about how amino acid sequence changes serve to alter a protein's conformational landscape, such that new regulatory strategies can be realized. The goal of this proposal is to illuminate how evolutionary trajectories, shaped by natural selection, provide access to new protein conformations, which facilitate the emergence of novel regulatory mechanisms. As a model system, we will study the evolution of regulation within the glucokinase (GCK) protein family. GCK is regulated by several mechanisms that require access to multiple conformations, and GCK is evolutionarily tractable ? its orthology is established and sequences from a range of species are available. We will use ancestral sequence reconstruction to resurrect ancestral versions of GCK and it's regulatory protein, GKRP. We will perform structural, biochemical and biophysical analyses of these proteins to illuminate the functional transitions that afford access to new protein conformations, which facilitate novel modes of regulation. Understanding the evolutionary origins of GCK's diverse regulatory processes will provide direct insight into the emergence of physiology and disease, as GCK is the primary glucose sensor in humans and a key determinant of glucose homeostasis in vertebrates. Fundamental questions that will be addressed in this proposal include: (1) How does a disordered polypeptide region evolve from an ordered precursor? (2) How do sequence changes expand the conformational landscape of a protein? (3) What events led to the emergence of allostery? (4) How does a new regulatory protein-protein interaction arise from two non-interacting ancestral proteins?